Background

Bioethanol production from lignocellulosic biomass, in particular xylose, is currently of great concern, given the abundance of this sugar in the world, because Saccharomyces cerevisiae, which is widely used for bioethanol production, is unable to naturally ferment xylose. The aim of this study was to obtain a novel yeast capable of stably producing ethanol from biomass containing xylose by protoplast fusion between S. cerevisiae and xylose-utilizing yeast.

Results

We describe a novel xylose-fermenting yeast strain, FSC1, developed for ethanol production by intergeneric hybridization between S. cerevisiae and Candida intermedia mutants by using a protoplast fusion technique. The characteristics of the FSC1 strain are reported with respect to xylose fermentation, morphology, gene, and protein expression. Mutation of the parental strains prior to protoplast fusion endowed the FSC1 strain with the ability to convert xylose to ethanol. Microscopic analysis confirmed that the parental and FSC1 strains produced spores in the potassium acetate medium. The FSC1 strain is uninucleate diploid, has a stable metabolism, and expresses proteins at a higher level than the parental strains. We found that FSC1 strain could stably achieve an ethanol yield of 0.38 g/g-substrate in fermentation of a mixture of glucose and xylose. In addition, the fermentation ability of FSC1was improved by successive chemical mutation, resulting in a higher ethanol yield of 0.42 g/g-substrate, corresponding to 82% theoretical yield.

Conclusions

The mutation-fusion technique we have described here is very useful for the development of intergeneric hybrids capable of xylose fermentation, and the FSC strains generated using this technique have the potential for industrial use in ethanol production from lignocellulosic biomass.

With the increasing appreciation of the problem of global warming, bioethanol has recently gained increasing attention as a renewable and carbon-neutral energy source. Bioethanol production through the fermentation of lignocellulosic biomass, in particular xylose, is currently of great concern, given the abundance of this sugar in wood and herbs. Given that Saccharomyces cerevisiae, which is widely used for bioethanol production, is unable to naturally ferment xylose, there is increasing investigation of its metabolic alteration to endow it with the ability to ferment xylose.

In general, fungal xylose fermentation initially requires two sequential reactions, namely, the conversion of xylose to xylitol, catalyzed by xylose reductase (XR), and the conversion of xylitol to xylulose, catalyzed by xylitol dehydrogenase (XDH). In a reaction catalyzed by xylulokinase (XK), xylulose is then phosphorylated to xylulose-5-phosphate before entering the pentose phosphate pathway (PPP). While S. cerevisiae is unable to ferment xylose because of the lack of XDH activity in the presence of glucose, it possesses the XYL2 gene, encoding XDH [1], as well as the homologous gene XKS1, encoding XK [2]–[4], and GRE3, encoding aldose reductase, which is closely related to XR [5]. In addition, the ability of S. cerevisiae to take up xylose through hexose transporter, and its aldose reductase activity, can be enhanced by chemical mutation and intensive screening on the basis of 2-deoxyglucose (DOG) tolerance [6].

The metabolic alteration of the yeast for ethanol production has been attempted through mutation, fusion, and recombination. Improvements in yeast metabolism have been reported by mutation and fusion between S. cerevisiae and Zygosaccharomyces fermentati[7], S. cerevisiae and Pichia stipitis[8], Kluyveromyces marxianus TS8-1 and TS87-8 [9], and S. cerevisiae and Candida shehatae[10]. During culture passages, however, some fusants were dissociated into segregants resembling the parental strains [8], and fusant offspring are almost completely sterile mainly because of the inability of the chromosomes of the partner genomes to pair or to recombine [11]. More recently, several attempts have been made to transfer specific genes for xylose utilization to S. cerevisiae by construction of recombinant strains (reviewed by Matsushika et al., ref [12]). The recombinants are expected to be practically applied for ethanol production from lignocellulose by overcoming such problems as redox imbalance in the initial step of xylose fermentation and reverse flux in glycolysis [13].

The xylose transporter of yeast Candida intermedia PYCC 4715, which grows equally well in xylose and glucose and has a high xylose transport capacity [14], has been functionally expressed in recombinant S. cerevisiae to increase its xylose uptake [15]. From these studies, we think that cell fusion between Saccharomyces and Candida strains may yield a strain which can take up and utilize xylose, as well as glucose, for ethanol production. Since cell fusion allows the transfer of complete segments of genomic DNA from parental yeasts, a fusant rich in genetic information could be obtained by protoplast fusion and stabilized by routine mutation and screening techniques. C. intermedia is nonpathogenic and safe for use. Consequently, C. intermedia has potential as a cell fusion partner with S. cerevisiae for the transfer of genes for xylose fermentation.

The aim of this study was to obtain a novel yeast capable of stably producing ethanol from biomass containing xylose by harnessing recent progress in intergeneric hybridization techniques with proteomic analysis. We developed a novel xylose-fermenting strain by intergeneric protoplast fusion between S. cerevisiae and C. intermedia strains altered, in advance, by mutation. The fusant obtained was subsequently characterized with respect to xylose fermentation, ethanol yield, morphology, and gene and protein expression.

Mutation of wild-type strains and fermentation by mutants

The mutant M2 strain improved in xylose uptake had been selected from diverse mutant colonies of S. cerevisiae grown on medium containing DOG as described in a previous study [6]. Since the M2 strain lacks the XDH activity, C. intermedia was used as a donor of xdh gene in cell fusion of this study. C. intermedia can originally take and metabolize xylose into ethanol, but its ability of ethanol production is not high. Therefore, it is important to use the C. intermedia mutant that has no ability for taking xylose upon the fermentation. As described in the Methods, C. intermedia was mutated using ethyl methane sulfonate (EMS) to obtain a strain in which xylose uptake was strongly suppressed, but which contained the xdh gene. DOG was used for screening DOG-sensitive mutants to surely repress the growth of parental m11 in regeneration of fused protoplast cells. We finally selected a mutant m11 strain considered to have the potential to endow the fusant with the ability to metabolize xylose when hybridized with the M2 strain by protoplast fusion, as described in the following hybridization.

Next, fermentation by m11 and its wild-type strains were investigated in MMGX medium. The results are shown in Figure 1, and include those of the M2 strains for reference. As initially intended, the m11 strain of C. intermedia consumed glucose but not xylose, while the wild-type strain utilized both glucose and xylose, with xylitol accumulating at high levels in the supernatant (Figure 1b). Ethanol production was low but glycerol was produced in both m11 and wild-type strains. On the other hand, the M2 strain of S. cerevisiae did take up xylose at a rate of 5.26 g-xylose/g-cell, while the original strain did at 1.58 g-xylose/g-cell, and produced more ethanol with less glycerol formation as shown in Figure 1a, also detailed in the previous study [6].

Before protoplast fusion, we investigated the sporulation of C. intermedia m11 using the potassium acetate (KAc) medium. Using the Wirtz-Conklin spore stain method, m11 cells after sporulation were stained greenish-blue by Brilliant Green, but were not stained pink by safranin, indicating no growth of the vegetative cell (Figure 2a and c). Spores appeared as spheres under scanning electron microscope (SEM) (Figure 2e), and were different in shape from the m11 cell, which was ellipsoidal, as described below. We confirmed that the m11 strain formed spores, as reported for the US patent of the parental C. intermedia (originally designated as a Kluyveromyces cellobiovorus) [16],[17]. Protoplasts of haploid cells obtained after sporulation of M2 and m11 strains were subjected to cell fusion and the regenerated cells were incubated in MMXDOG medium. Since the M2 strain is tolerant to DOG but is unable to use xylose, and the m11 strain is sensitive to DOG inhibition, only heterogenic fusants could form colonies in the medium. The suppression of xylose uptake in the m11 strain by mutation also allowed the selection of a target fusant without growth of the m11 strain in medium containing xylose as a carbon source.

Figure 2

Spore formations ofC. intermediam11 (left) and FSC1 (right) strains in KAc medium. Microscopic observation with brilliant green staining before (a, b) and after (c, d) swelling the cell by soaking in saline solution, and SEM of the spores (e, f).

A target strain that first appeared was selected from three colonies formed in MMXDOG medium and named FSC1, as a fusant between S. cerevisiae M2 and C. intermedia m11. Brilliant Green staining and SEM observation confirmed that the FSC1 strain produced spores in KAc medium (Figure 2b, d and f).

Colony formations of FSC1, M2 and m11 strains were investigated in YMG, YMFDOG and YMXDOG medium (Figure 3a, b and c, respectively). All strains grew in YMG, whereas FSC1 and M2 strains grew in YMFDOG, and only FSC1 strain grew in YMXDOG. These data indicated that the FSC1 cells were an intergeneric hybrid of the M2 and m11 cells. To investigate the possibility of normal mating of the partner strains instead of their haploid fusion for hybridization, we next attempted colony formation by mixing cultures of M2 and m11 strains cultivated in advance. As shown in Figure 3d, mixed cultures of M2 and m11 strains failed to form colonies in MMXDOG medium, indicating that the FSC1 strain was obtained by cell fusion, rather than normal mating between the M2 and m11 strains.

Figure 3

Colony formations of the M2, m11, and FSC1 strains on various YM agar media. (a) Colony formations of each strain on YMG, (b) YMFDOG, (c) YMXDOG, and (d) the mixed culture of M2 and m11 strains cultivated in advance and the FSC strain as a control on MMXDOG at 30°C for 3 days.

Next, the morphology of the M2, m11 and FSC1 strains was microscopically examined (Figure 4a, b and c, respectively). The M2 cells appeared ellipsoidal, while m11 cells were similar in shape but smaller in size. The FSC1 cells also appeared ellipsoidal and were much larger than the parental cells, with larger cells having a long axis of approximately 10 μm. 4′,6-diamidino-2-phenylindole (DAPI) stain confirmed that all FSC1 cells were uninucleate as shown by the arrow in Figure 4d.

Metabolism by xylose-fermenting fusant FSC1

We investigated fermentation by the FSC1 strain in MMGX medium, as conducted for the parental strains. Both glucose and xylose were completely fermented and ethanol production was approximately 2.7-fold higher than in the M2 strain (Figures 5a and 1a). The FSC1 strain produced some xylitol, but much less glycerol than the m11 strain. The ethanol yield for the FSC1 strain (0.38 g/g-substrate) was high compared to the wild-type and mutant strains (0.10 and 0.14 g/g-substrate in S. cerevisiae and 0.07 and 0.03 g/g-substrate in C. intermedia, respectively) (Figure 5b). Fermentation characteristics of the FSC1 strain were quantitatively summarized in Table 1.

Figure 5

Fermentation by the fusant FSC1 strain using glucose and xylose as carbon sources. (a) Ethanol and other metabolites production by the FSC1 strain, (○) glucose, (□) xylose, (●) ethanol, (■) xylitol, (Δ) glycerol. (b) Ethanol yields of the FSC1 and parental strains. (c) Fermentation stability of the FSC1 strain.

Stability of metabolism is also concern for strains altered by mutation and fusion. As shown in Figure 5c, the FSC1 strain showed a high stability in both ethanol production and xylose consumption over 14 generations.

Next, we investigated the amino acid requirements of the FSC1, M2 and m11 strains, using seven amino acids in MMG medium (Table 2). The M2 strain grew in MMG while the m11 strain did not, although both strains grew in the enriched medium. The FSC1 strain grew in MMG, indicating the strain was prototrophic, as was observed for the M2 strain. On the other hand, the m11 strain was auxotrophic absolutely for uracil and relatively for histidine.

Mutation is often used to generate improved yeast strains [18]. We tried to improve the fermentation ability of the FSC1 strain by mutating twice using EMS in the manner described for the parental strains in Methods. The FSC1 mutant obtained showed 0.42 g/g-substrate in ethanol yield, 10% higher than by the FSC1 strain, as shown in Table 1. A xylose consumption rate was also 6 times higher, improved from 0.18 to 1.07 g/g-cell.h.

mRNA and total protein expression

We used the reverse transcription polymerase chain reaction (RT-PCR) to analyze the mRNA expression levels of genes related to xylose fermentation (xr, xdh, xk, and adh1). As shown in Figure 6a, analysis of the FSC1 strain indicated that xr and xdh were transferred from C. intermedia (Cdxr and Cdxdh), with reduced expression of gre3 from S. cerevisiae (Scgre3 corresponding to xr), while xk and adh1 were transferred from S. cerevisiae (Scxks1 corresponding to xk, and Scadh1). Expression levels of Cdxr, Cdxdh, Scxks1 and Scadh1 in the FSC1 strain were higher than those in the parental strains.

Figure 6

Expressions of genes for xylose fermentation analyzed by RT-PCR and proteins by 2D-PAGE. (a) Genes for xylose fermentation by S. cerevisiae M2, C. intermedia m11, and FSC1 strains microaerobically grown on glucose and xylose as carbon sources. (b) Protein spots of each strain (100 μg protein extracts on the gels), in which the horizontal axis is the isoelectric focusing dimension from pH3 to pH10 and the vertical axis is 15% (w/v) polyacrylamide gel dimension from 11 kDa to 250 kDa.

With regard to total protein expression analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), most of the protein spots detected in the M2 and m11 strains were present at higher levels in the FSC1 strain (Figure 6b). Based on a search using Mascot, two of the proteins were considered to match to XR (score = 72, required score > 43, p < 0.05) and glyceraldehyde-3-phosphate dehydrogenase (TDH) (score = 130, required score > 59, p < 0.05).

Fusants are generally less stable in metabolism because of the loss of non-homologous genes and chromosomes in chromosome segregations. To overcome this problem, we used haploid cells formed under suppression of mating by exogenous α factor in the M2 strain [19], anticipating a similar effect in the m11 strain, and performed electrofusion to attain more stable uninucleate polyploids [20] after chemical fusion using polyethylene glycol (PEG). Exogenous α factor inhibits mating when present in excess [19], though it is reported that prior activation of cells by α factor induces nuclear fusion [21]. In addition, we employed C. intermedia as a donator of the genes for xylose fermentation on the basis of a study reporting that genes and proteins necessary for xylose fermentation from C. intermedia can be functionally expressed in recombinant S. cerevisiae[15].

The FSC1 strain possesses high and stable rates of xylose fermentation and ethanol production from a substrate containing glucose and xylose. We consider that the mutation of the parental strains enabled their fusion to transfer genes for xylose fermentation of C. intermedia and for ethanol production of S. cerevisiae. Since the FSC1 strain was apparently uninucleate (Figure 4d), we confirmed that our mutation-fusion technique resulted in nuclear fusion of protoplasts, which is desirable since homocaryons are metabolically more stable than heterocaryons. Since the FSC1 strain produces spores in KAc medium for sporulation (Figure 2b, d and f), we believe that the FSC1 strain is diploid, resulting from fusion between M2 and m11 haploids. This is not contradictory to previous studies reporting that several Candida species have MTL loci with two idiomorphs, namely a and α, which are mating type-like loci similar to MATa and MATα of S. cerevisiae[22], although Candida is a large and heterogeneous taxon.

All genes necessary for xylose metabolism xr, xdh, and xk (xks1) were expressed at a higher level in FSC1 than in the parental strains (Figure 6a). Moreover, the level of total protein expression in FSC1 was also higher than a simple summation of the parental strains when the same amount of crude cell extracts were loaded on gel (Figure 6b), most likely due to the activation in FSC1 of numerous metabolic pathways during fermentation. RT-PCR and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) analysis indicated that XR derived from C. intermedia m11, and TDH from S. cerevisiae M2, were both expressed in FSC1. Since TDH is required for glycolysis and, by extension, cell viability [23], the expression of TDH in the FSC1 strain is important to convert xylose to ethanol via glycolysis. These results indicate that the FSC1 strain is an intergeneric hybrid between S. cerevisiae M2 and C. intermedia m11, a fact supported by the observation that normal mating of the parental strains did not occur (Figure 3d).

The metabolic properties of the FSC1 strain indicate that it maintains the redox balance required for fermentation inside the cell. Redox imbalance, which is thought to be caused by coenzyme specificity differences between heterogenic XR (with NADPH) and XDH (with NAD+) enzymes, is a major cause of xylitol accumulation inside and outside the cell, resulting in the reduction of ethanol yield [24]–[26]. Since cell fusion allows the transfer of complete segments of genomic DNA from parental cells, a fusant will be rich in genetic information. We suggest that the higher expression of proteins in the FSC1 strain is not caused simply by the activation of metabolic pathways (PPP) for xylose fermentation, but by the presence of overall metabolic (glycolysis and PPP) regulation to maintain the redox balance for fermentation inside the cell. This is a key point behind the improved xylose utilization, which was supposed from the protein level.

Although fermentation was conducted under microaerobic conditions, undesirable byproducts as glycerol and acetate were not produced. As summarized in Table 1, the ethanol yield and production rate of the FSC1 strain were 0.38 g/g-substrate, corresponding to 75% theoretical yield, and 0.33 g/L · h in fermentation of the mixture of glucose and xylose, respectively. In addition, the fermentation ability of FSC1was further improved by successive mutation, achieving a higher ethanol yield (0.42 g/g-substrate). The ethanol yields of the FSC strains are comparable to 0.34 g/g-substrate of engineered strains of the recombinant S. cerevisiae TMB3400, generated by introducing the gene for xylose transporter from C. intermedia[27], in a mixture of xylose and glucose, and 0.05-0.46 g/g-substrate in fermentation of xylose as a sole carbon source by various recombinant S. cerevisiae strains [12]. Although the xylose consumption rate (1.07 g/g-cell. h, i.e., 7.1 mmol/g-cell. h) of FSC1 mutant was lower than those with previous reports such as the recombinant S. cerevisiae TMB3400 [27], the substrate was completely consumed within 30 h, shorter than a normal reaction time 48 h in ethanol fermentation for practical use. We believe that the mutation-fusion technique developed in this study is applicable for metabolic alteration of ethanol producing yeasts as a diverse method from recombination.

We developed a novel xylose-fermenting yeast strain, FSC1, for ethanol production by intergeneric hybridization between Saccharomyces cerevisiae and Candida intermedia mutants by using a protoplast fusion technique. The fermentation ability of the FSC1 strain was further improved by chemical mutation. The mutation-fusion technique we have described is useful for the development of an intergeneric fusant capable of xylose fermentation. The FSC strains obtained by this technique hold the potential for ethanol production from globally abundant lignocellulosic biomass.

Strains and cultivation media

Wild-type yeast strains S. cerevisiae NBRC 2114 and C. intermedia NBRC 10601 were obtained from the NITE Biological Resource Center (NBRC) at the National Institute of Technology and Evaluation (NITE) in Tsukuba, Japan. C. intermedia NBRC 1060 was originally designated as a K. cellobiovorus strain that was reported to be capable of producing ethanol from xylose in 1984 [16],[17] but was later classified as a neotype of C. intermedia (Ciferri & Ashford) Langeron et Guerra [28]. S. cerevisiae is taxonomically classified into Saccharomycetaceae in family and C. intermedia NRRL Y-981 belongs to the Metschnikowia clade [29]. Because S. cerevisiae and C. intermedia belong to different taxonomical families, experiments on cell fusion were conducted taking the containment measures confirmed by the competent minister under the Act on the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms (Act No. 97 of 2003, Japan).

All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA), except where mentioned otherwise.

Mutation and screening

To obtain a variety of species of different phenotypes for screening target mutants, S. cerevisiae and C. intermedia strains were individually mutated using EMS (Wako Pure Chemicals, Ltd., Osaka, Japan) as described in our previous study [6]. The strain was grown at 30°C for 6 h in 50 mL of YM medium in 1 L of distilled water. Next, a 1-mL aliquot of the cell suspension was transferred to a 1.5-mL tube. After centrifugation, the cell pellets were washed twice with cold 0.1 M sodium phosphate buffer (pH 7.0), then suspended in 1 mL of the same buffer. EMS (30 μL, purity ≥ 98%) was added to the suspension, giving a final volume of EMS at nearly 3% (v/v). The tube was incubated at 30°C on a roller shaker for 60 min. The reaction was stopped by adding 10% (w/v) sterile sodium thiosulfate solution at a final concentration of 1% (v/v), and the suspension was centrifuged at 5,000 × g for 1 min. After removal of the supernatant, 1 mL of 10% (w/v) sterile sodium thiosulfate solution was added to the pellet, and the suspension was mixed and incubated at room temperature for 15 min to completely terminate the reaction.

After mutation, the mutant cells were washed with 0.1 M sodium phosphate buffer (pH 7.0) three times, and 100-μL aliquots of the suspension were spread on YMFDOG containing 3 g/L DOG. Replica plates were also prepared on YMG, and the plates were incubated at 30°C for colony formation. To screen a DOG-tolerant mutant (named the M2 strain) from S. cerevisiae strain, colonies that appeared were replicated on YMFDOG containing 5 g/L DOG and then screened after incubation at 30°C for 3 days. To screen a DOG-sensitive mutant (named the m11 strain) from C. intermedia, colonies that were unable to grow on YMFDOG containing 3 g/L DOG, were selected from the replica plates. Growth of both mutant strains was evaluated on YMG.

Seed preparation and fermentation

Seed preparation for fermentation was performed in two steps. In the first step, a single colony of the yeast was inoculated into 100 mL of YMG liquid medium in a 500-mL flask after autoclaving at 122°C for 20 min, and then incubated at 30°C overnight on a shaker at 150 rpm. In the second step, 10 mL of the first seed were transferred in quadruplicate to 100 mL of YMGX liquid medium in a 500-mL flask, which was then incubated at 30°C for 24 h on a shaker at 150 rpm. After collecting and washing twice with sterile phosphate-buffered saline (PBS), cells were resuspended in PBS in a minimal volume (50 ml).

Fermentation was started by adding 50 mL of cell suspension containing 1.3–1.5 g-dried weight (DW) of cells to 950 mL of MMGX medium in a jar fermenter. 5 N NaOH was used to maintain pH 5 in the culture. To maintain microaerobic conditions, air was pumped through a sterilized membrane filter into the reactor to maintain 5% dissolved oxygen under the air-saturated condition. Levels of glucose, xylose, xylitol, glycerol, and ethanol in the culture medium were quantified by high performance liquid chromatography (HPLC) as described in the previous study [6]. Stability of fermentation by fusant cells was confirmed in both ethanol production and xylose consumption over 14 generations. Generation in this context refers to the culture of glycerol stock cells obtained from one single colony appearing on appropriate cultivation agar YMGXDOG.

Gene and total protein expression analyses

Cells were harvested from the jar fermenter directly after depletion of the carbon sources, washed with cold sterile water twice and then freeze-dried. Freeze-dried cells (100 mg) were suspended in 250 μL Yeast protein extraction reagent (Y-PER) supplemented with 5 μL protease inhibitor (Wako Pure Chemicals, Ltd., Osaka, Japan), incubated for 60 min, then centrifuged at 14,000 × g for 10 min. To remove excess salts, the supernatant was passed through a desalting column Bio-Gel P-6 (BioRad Lab., Inc., Hercules, CA) buffered with 10 mM Tris/HCl (pH7.4), and the eluate was used as a protein mixture sample for total protein analyses.

For total protein expression, the protein extract of each strain was analyzed by 2D-PAGE as previously reported [32]. Isoelectric focusing (IEF) electrophoresis of desalted protein samples was performed using IPG ReadyStrip pH3-10 NL (BioRad Lab., Inc.) conditioned in Protean IEF system (BioRad Lab., Inc.). We prepared the crude cell extracts adjusted to 100 μg respectively from M2, m11 and FSC1 strains, under the same procedure. After treatment in an alkylation solution containing 100 mM iodoacetate, 6 M urea, 2% (w/v) sodium dodecyl sulfate (SDS) and 20% (v/v) glycerol in 0.375 M Tris/HCl (pH 8.8), the IPG strip was applied to a 15% non-gradient SDS-PAGE electrophoresis. Finally, the developed gel was stained using a sensitive colloidal Coomassie G-250 solution to observe significant changes of total protein expression.

Protein spots were identified by MALDI-TOF/TOF analyses. Spots were enzymatically digested in a manner similar to that previously described [33] using modified porcine trypsin (Promega Corp., Madison, WI). Gel pieces were washed with 50% (v/v) acetonitrile to remove SDS, salt, and stain. Washed and dehydrated spots were then vacuum-dried to remove solvent and rehydrated with trypsin (8–10 ng/μL) solution in 50 mM ammonium bicarbonate at pH 8.7 and incubated for 8–10 h at 37°C. The samples were analyzed using an Applied Biosystems 4700 proteomics analyzer with TOF/TOF ion optics (Genomine Inc. Pohang, Korea). Sequence tag searches were performed using Mascot search (http://www.matrixscience.com).

Microscopic observation

For microscopic observation of sporulation, each strain was sporulated using KAc agar containing 10 g/L KAc and 20 g/L Bacto agar for 7 days at 30°C in the manner described earlier. After incubation, spores were removed from the surface of the agar medium by washing with 0.05% Tween 80 in saline solution. The suspension was centrifuged at 3,000 × g for 10 min at 4°C. The supernatant was transferred into fresh tube and kept at 4°C until use. To confirm sporulation using the Wirtz-Conklin spore staining technique [34], the spores were strained with 5% brilliant (malachite) green (dye for staining spores) solution on a slide, heated with a Bunsen flame for 5 min and washed with MilliQ water, then counterstained with 0.5% safranin (dye for staining vegetative cells) solution for 1 min. Staining was conducted before and after swelling the cell by soaking in saline solution for a few days. After drying, the slide was observed under a light microscope. SEM observation was also performed to confirm the morphology of ascospores from each strain. The spores were fixed overnight at 4°C with 0.1% (vol./vol.-PBS buffer) glutaraldehyde, washed three times with PBS buffer, dehydrated in an ethanol series, then dried at room temperature. After coating with Pt-Pd using a sputter coater (Hitachi E102 Ion Sputter, Hitachi, Tokyo, Japan) for 2 min at DC20 mA, spore samples were observed with SEM (Hitachi S-4700 Type II, Hitachi, Tokyo, Japan) at 10 kV.

PK was a researcher of the Laboratory of Water Environment and Bioenergy at Meisei University, when the study was conducted from 2010–2012. ST is a professor of the laboratory and was a representative of the study.

Acknowledgments

This research was financially supported by the Environment Research and Technology Development Fund (K22027, K2339, and K2411) from Ministry of the Environment, Japan. We thank Mr. Kazuo Taku and the staff of the Collaborative Research Center of Meisei University for their assistants on measurements and analyses.

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Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

PK carried out the experiments and drafted the manuscript. ST conceived and designed the study as a research representative, and completed the manuscript. Both authors read and approved the final manuscript.

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